Sunggi Heu

A comprehensive expression analysis of the WRKY gene superfamily in rice plants during defense response

To understand the transcriptional regulatory mechanism of host genes during the activation of defense responses in rice, we isolated WRKY transcription factors whose expressions were altered upon attack of the fungal pathogen Magnaporthe grisea, the causal agent of the devastating rice blast disease. A systematic expression analysis of OsWRKYs (Oryza sativa L. WRKYs) revealed that among 45 tested genes the expression of 15 genes was increased remarkably in an incompatible interaction between rice and M. grisea. Twelve of the M. grisea-inducible OsWRKY genes were also differentially regulated in rice plants infected with the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In experiments with defense signaling molecules, the expression of two genes, OsWRKY45 and OsWRKY62, was increased in salicylic acid (SA)-treated leaves and the expression of three genes, OsWRKY10, OsWRKY82, and OsWRKY85 was increased by jasmonic acid (JA) treatment. OsWRKY30 and OsWRKY83 responded to both SA- and JA treatments. The expression profiles suggest that a large number of WRKY DNA-binding proteins are involved in the transcriptional activation of defense-related genes in response to rice pathogens.

Combined effect of ultrasound and organic acids to reduce Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on organic fresh lettuce.

This study was performed to compare the effectiveness of individual treatments (ultrasound and organic acids) and their combination on reducing foodborne pathogens on organic fresh lettuce. Lettuce leaves were inoculated with a cocktail of three strains each of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes and treated with ultrasound (40 kHz) alone, organic acids (0.3, 0.5, 0.7, 1.0, and 2.0%--malic acid, lactic acid, and citric acid) alone and combined with ultrasound and organic acids for 5 min. For all 3 pathogens, the combined treatment of ultrasound and organic acids resulted in additional 0.8 to 1.0 log reduction compared to individual treatments, without causing significant quality change (color and texture) on lettuce during 7 day storage. The maximum reductions of E. coli O157:H7, S. Typhimurium, and L. monocytogenes were 2.75, 3.18, and 2.87 log CFU/g observed after combined treatment with ultrasound and 2% organic acid for 5 min, respectively. Our results suggest that the combined treatment of ultrasound with organic acids was effective at increasing pathogen reduction compared to individual treatments without significantly affecting quality, and demonstrates its potential as a novel method to increase the microbial safety on organic fresh lettuce.

Molecular cloning and mRNA expression analysis of a novel rice (Oryzasativa L.) MAPK kinase kinase, OsEDR1, an ortholog of ArabidopsisAtEDR1, reveal its role in defense/stress signalling pathways and development

Mitogen-activated protein kinase (MAPK) cascade(s) is important for plant defense/stress responses. Though MAPKs have been identified and characterized in rice (Oryza sativa L.), a monocot cereal crop research model, the first upstream component of the kinase cascade, namely MAPK kinase kinase (MAPKKK) has not yet been identified. Here we report the cloning of a novel rice gene encoding a MAPKKK, OsEDR1, designated based on its homology with the Arabidopsis MAPKKK, AtEDR1. OsEDR1, a single copy gene in the genome of rice, encodes a predicted protein with molecular mass of 113046.13 and a pI of 9.03. Using our established two-week-old rice seedling in vitro model system, we show that OsEDR1 has a constitutive expression in seedling leaves and is further up-regulated within 15 min upon wounding by cut, treatment with the global signals jasmonic acid (JA), salicylic acid (SA), ethylene (ethephon, ET), abscisic acid, and hydrogen peroxide. In addition, protein phosphatase inhibitors, fungal elicitor chitosan, drought, high salt and sugar, and heavy metals also dramatically induce its expression. Moreover, OsEDR1 expression was altered by co-application of JA, SA, and ET, and required de novo synthesized protein factor(s) in its transient regulation. Furthermore, using an in vivo system we also show that OsEDR1 responds to changes in temperature and environmental pollutants-ozone and sulfur dioxide. Finally, OsEDR1 expression varied significantly in vegetative and reproductive tissues. These results suggest a role for OsEDR1 in defense/stress signalling pathways and development.

Prevalence and genetic diversity of Enterobacter sakazakii in ingredients of infant foods

Various food samples in Korea were examined for the presence of Enterobacter sakazakii. Dried shrimp had the highest contamination rate among the examined dried fish products. E. sakazakii isolates were confirmed using an API 20E kit and a polymerase chain reaction (PCR) targeting the rDNA operons. The isolates were subtyped by pulsed-field gel electrophoresis (PFGE) using XbaI to elucidate the genetic diversity of the organisms. Ten pulsotypes were identified using PFGE and 22 types were identified from the random amplified polymorphism DNA (RAPD) assay. An antibiotic resistance test was performed by disk diffusion assay using eight antibiotics: nalidixic acid, ciprofloxacin, chloramphenicol, ampicillin, tetracycline, gentamicin, kanamycin, and cephalothin. Most of the E. sakazakii isolates were resistant to ampicillin or cephalothin but susceptible to the other antibiotics. The analysis of E. sakazakii isolates using PFGE, RAPD, and the antibiotic resistance test identified 18 composite types from 113 isolates, suggesting diverse sources of contamination.

Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7.

ABSTRACT Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae. In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 × 10−8 ml/min and 1.91 × 10−8 ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10−2 CFU/ml for S. Typhimurium and 4.58 × 10−5 CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.

gly gene cloning and expression and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines 8ra.

ABSTRACT Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60°C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes,glyA and glyB, encoding the 39- and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences ofglyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with eitherglyA or glyB separately did not show the bacteriocin activity.

Identification of a novel pathogen-induced gene encoding a leucine-rich repeat protein expressed in phloem cells of Capsicum annuum

The CALRR1 gene, expressed in pepper leaves following infection by Xanthomonas campestris pv. vesicatoria, encodes a secreted leucine-rich repeat (LRR) with five tandem repeats of a 24-amino-acid LRR motif. Northern blot analyses revealed that CALRR1 is not constitutively expressed in pepper plants, but is strongly induced upon the infection by X. campestris pv. vesicatoria, Phytophthora capsici, Colletotrichum coccodes and Colletotrichum gloeosporioides on leaves. CALRR1 was not systemically induced in upper leaves by bacterial infection. The inoculation of bacterial live cells, and treatment with dead cells and culture filtrates of pathogenic or nonpathogenic bacteria triggered the accumulation of CALRR1 transcripts. Treatment with signaling molecules, including salicylic acid (SA), ethylene (ET), methyl jasmonate (MeJA), dl-beta-amino-n-butyric acid (BABA) and benzothiadiazole (BTH), did not activate the transcription of the CALRR1 gene, indicating that CALRR1 expression is not regulated by defense signaling pathways activated by these molecules. CALRR1 was induced by treatment with high salinity, abscisic acid (ABA) and wounding, but not by drought and cold stress. An in situ hybridization study showed that CALRR1 mRNA was localized in phloem tissues of leaves, stems, and green fruits of pepper plants during the pathogen infection and ABA exposure. The location characteristics and the spatio-temporal expression pattern of CALRR1 suggest that it may play a role in protecting phloem cells against biotic and abiotic stresses affecting phloem function.

Characterization of LysB4, an endolysin from the Bacillus cereus-infecting bacteriophage B4

BackgroundBacillus cereus is a foodborne pathogen that causes emetic or diarrheal types of food poisoning. The incidence of B. cereus food poisoning has been gradually increasing over the past few years, therefore, biocontrol agents effective against B. cereus need to be developed. Endolysins are phage-encoded bacterial peptidoglycan hydrolases and have received considerable attention as promising antibacterial agents.ResultsThe endolysin from B. cereus phage B4, designated LysB4, was identified and characterized. In silico analysis revealed that this endolysin had the VanY domain at the N terminus as the catalytic domain, and the SH3_5 domain at the C terminus that appears to be the cell wall binding domain. Biochemical characterization of LysB4 enzymatic activity showed that it had optimal peptidoglycan hydrolase activity at pH 8.0-10.0 and 50°C. The lytic activity was dependent on divalent metal ions, especially Zn2+. The antimicrobial spectrum was relatively broad because LysB4 lysed Gram-positive bacteria such as B. cereus, Bacillus subtilis and Listeria monocytogenes and some Gram-negative bacteria when treated with EDTA. LC-MS analysis of the cell wall cleavage products showed that LysB4 was an L-alanoyl-D-glutamate endopeptidase, making LysB4 the first characterized endopeptidase of this type to target B. cereus.ConclusionsLysB4 is believed to be the first reported L-alanoyl-D-glutamate endopeptidase from B. cereus-infecting bacteriophages. The properties of LysB4 showed that this endolysin has strong lytic activity against a broad range of pathogenic bacteria, which makes LysB4 a good candidate as a biocontrol agent against B. cereus and other pathogenic bacteria.

Prevalence and genetic diversity of Bacillus cereus in dried red pepper in Korea.

Bacillus cereus is a foodborne spore-forming bacterial pathogen that is ubiquitous in the natural environment. Infections with this pathogen manifest as diarrheal or emetic types of food poisoning. In this study, 140 samples of dried red pepper purchased in Korea were assayed for the presence of B. cereus according to the U.S. Food and Drug Administration standard culture method. A multiplex PCR assay was developed for the rapid confirmation of B. cereus as an alternative to conventional biochemical confirmation tests. The genetic diversity of B. cereus isolates was investigated using a random amplified polymorphic DNA (RAPD) assay. B. cereus was found in 84.3% of the dried red pepper samples, with an average concentration of 1.9 x 10(4) CFU/g. B. cereus could be detected and distinguished from B. thuringiensis in the multiplex PCR assay by using the BCFW1 plus BCrevnew and the K3 plus K5 primer sets designed to detect the gyrB gene of B. cereus and B. thuringiensis and the cry gene of B. thuringiensis. A RAPD assay using the OPG 16 and MUP 3 primers was used to successfully distinguish among isolates, thus elucidating the genetic diversity of B. cereus isolates. The discriminating ability of the OPG 16 primer (142 types) was about threefold higher than that of MUP 3 (52 types) in the RAPD assay.

Receptor Diversity and Host Interaction of Bacteriophages Infecting Salmonella enterica Serovar Typhimurium

Background Salmonella enterica subspecies enterica serovar Typhimurium is a Gram-negative pathogen causing salmonellosis. Salmonella Typhimurium-targeting bacteriophages have been proposed as an alternative biocontrol agent to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated. Methodology/Principal Findings Twenty-five Salmonella phages were isolated and their receptors were identified by screening a Tn5 random mutant library of S. Typhimurium SL1344. Among them, three types of receptors were identified flagella (11 phages), vitamin B12 uptake outer membrane protein, BtuB (7 phages) and lipopolysaccharide-related O-antigen (7 phages). TEM observation revealed that the phages using flagella (group F) or BtuB (group B) as a receptor belong to Siphoviridae family, and the phages using O-antigen of LPS as a receptor (group L) belong to Podoviridae family. Interestingly, while some of group F phages (F-I) target FliC host receptor, others (F-II) target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain. Conclusions/Significance In this report, three receptor groups of 25 newly isolated S. Typhimurium-targeting phages were determined. Among them, two subgroups of group F phages interact with their host receptors in different manner. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for Salmonella phages and will inform development of optimal phage therapy for protection against Salmonella.